The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells.

BACKGROUND: The human T-cell leukemia virus type 1 (HTLV-1) Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. RESULTS: To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. CONCLUSION: These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.

Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in HTLV-1 Tax expressing cells.

Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) are associated with Human T-cell lymphotropic virus type 1 (HTLV-1) infection. The viral transactivator, Tax is able to mediate the cell cycle progression by targeting key regulators of the cell cycle such as p21/waf1, p16/ink4a, p53, cyclins D1-3/cdk complexes, and the mitotic spindle checkpoint MAD apparatus, thereby deregulating cellular DNA damage and checkpoint control. Genome expression profiling of infected cells exemplified by the development of DNA microarrays represents a major advance in genome-wide functional analysis. Utilizing cDNA microarray analysis, we have observed an apparent opposing and paradoxical regulatory network of host cell gene expression upon the introduction of DNA damage stress signal. We find the apparent induction of cell cycle inhibitors, and pro- as well as anti-apoptotic gene expression is directly linked to whether cells are at either G1, S, or G2/M phases of the cell cycle. Specifically, a G1/S block is induced by p21/waf1 and p16/ink4a, while pro-apoptotic expression at S, and G2/M is associated with caspase activation, and anti-apoptotic gene expression is associated with up regulation of Bcl-2 family member, namely bfl-1 gene. Therefore, the microarray results indicating expression of both pro- and anti-apoptotic genes could easily be explained by the particular stage of the cell cycle. Mechanism and the functional outcome of induction for both pathways are discussed.

Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics.

Human immunodeficiency virus type 1 (HIV-1) can infect quiescent cells; however, viral production is restricted to actively proliferating cells. Recent evidence has indicated that HIV-1 viral proteins, Vpr and Tat, perturb the cell cycle to optimize HIV-1 replication. Vpr arrests the cell cycle at G2 by inactivating the cyclin B/cdk1 complex. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation (i.e. the cdk inhibitor p21/waf1) and associating with cyclin/cdk complexes (i.e. cyclin E/cdk2, cyclin H/cdk7, and cyclin T/cdk9). These studies indicate the importance of host cellular factors, such as cyclin/cdk complexes, in regulating HIV-1 replication and therefore represent novel targets for antiviral therapeutics. Recently, the efficacy of pharmalogical cdk inhibitors (PCIs) in abrogating viral replication has been under development. To date there are 25-30 PCIs that have been synthesized against known cdks, several of which have been shown to inhibit HIV-1 and other AIDS-associated viruses in vitro and in vivo. Targeting these critical cyclin/cdk complexes needed for viral propagation may solve the problems inherent in current HAART therapy, including the emergence of drug-resistant viruses. Thus, PCIs have the potential to become novel therapeutic antiviral drugs that can inhibit HIV-1 transcription and opens the possibility of new avenues of treatment.

Inhibition of HTLV-1 transcription by cyclin dependent kinase inhibitors.

HTLV-1 is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), where viral replication and transformation are largely dependent upon modification of regulatory and host cell cycle proteins. The mechanism of HTLV-1 transformation appears to be distinct from that of many known chronic or acute leukemia viruses and is related to the viral activator Tax. Here we show that cyclin E, can associate tightly with the coactivator p300 and Pol II complex in HTLV-1 infected cells. The cyclin E associated complex is kinase active and phosphorylates the carboxy terminal domain of RNA Pol II. More importantly, p21/Waf1, a well-known cdk inhibitor at the G1/S border, inhibits transcription of HTLV-1 in both transfections and in in vitro transcription assays. Finally, specific cdk chemical inhibitors, functionally similar to cellular cdkIs, such as p21/Waf1 which inhibits cyclin E/cdk2 activity, also inhibit transcription of the HTLV-1 promoter. In particular, Purvalanol A, with an IC50 of 0.035 microm inhibits activated, but not basal transcription, as well as HTLV-1 infected cells. Collectively, the role of cyclin E/cdk2 in HTLV-1 infected cells and its involvement in RNA Pol II phosphorylation is discussed.

Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals.

BACKGROUND: Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator. RESULTS: Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins. CONCLUSIONS: We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals.

Enhancement of nuclear factor-kappa B acetylation by coactivator p300 and HIV-1 Tat proteins.

Nuclear factor (NF)-kappaB transcription factors are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. Transcription of the human immunodeficiency virus type 1 (HIV-1) genome depends on the intracellular environment where the integrate viral DNA is regulated by a complex interplay among viral regulatory proteins, such as Tat, and host cellular transcription factors, such as NF-kappaB, interacting with the viral long terminal repeat region. CBP (CREB-binding protein) and p300, containing an intrinsic histone acetyltransferase (HAT) activity, have emerged as coactivators for various DNA-binding transcription factors. Here, we show that the p50 subunit as well as the p50/p65 of NF-kappaB, and not other factors such as SP1, TFIIB, polymerase II, TFIIA, or p65, can be acetylated by CBP/p300 HAT domain. Acetylation of p50 was completely dependent on the presence of both HAT domain and Tat proteins, implying that Tat influences the transcription machinery by aiding CBP/p300 to acquire new partners and increase its functional repertoire. Three lysines, Lys-431, Lys-440, and Lys-441 in p50 were all acetylated in vitro, and a sequence similarity among p50, p53, Tat, and activin receptor type I on these particular lysines was observed. All proteins have been shown to be acetylated by the CBP/p300 HAT domain. Acetylated p50 increases its DNA binding properties, as evident by streptavidin/biotin pull-down assays when using labeled NF-kappaB oligonucleotides. Increased DNA binding on HIV-1 long terminal repeat coincided with increases in the rate of transcription. Therefore, we propose that acetylation of the DNA binding domain of NF-kappaB aids in nuclear translocation and enhanced transcription and also suggest that the substrate specificity of CBP/p300 can be altered by small peptide molecules, such as HIV-encoded Tat.